Development of the bitterling,Paracheilognathus himantegus (Cyprinidae), with a note on minute tubercles on the skin surface

The development of the eggs and larvae and minute tubercles on the skin surface ofParacheilognathus himantegus larvae were observed. The egg began to hatch approximately 68 hours after insemination and the larvae reached the free-swimming stage 23 days after hatching at water temperature of 22±1°C. The larval development and minute tubercles on the skin surface of this species were similar to those ofAcheilognathus lanceolata, A. limbata, A. signifer andTanakia tanago. However, the shape of the ripe eggs ofP. himantegus differed from those of the four species. As regards the shape of eggs, there was a common characteristic amongP. himantegus, Rhodeus uyekii andA. limbata from Korea. As regards larval development,P. himantegus had two characters also found inRhodeus. These facts seem to suggest thatP. himantegus is closely related toA. lanceolata, A. limbata, A. signifer andT. tanago but is more specialized than these four species, except forA. limbata from Korea.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.     

Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum

Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.     

The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity

We have identified a minimum functional domain of human thrombomodulin for anticoagulant activity using deletion analysis. Four mutants were constructed by site-directed deletion mutagenesis to delete one or more epidermal growth factor (EGF)-like structures from the domain of human thrombomodulin containing six repeated EGF-like structures. These deletion mutants were expressed transiently in COS-1 cells, and their protein C-activating cofactor activities in the culture medium were examined. One mutant protein, E456, which contains the fourth, fifth, and sixth EGF-like structures expresses apparent cofactor activity. However, neither E456-N24 (24 NH2-terminal-residue deletion) nor E456-C16 (16 COOH-terminal-residue deletion) have cofactor activity. E456 was partially purified and its anticoagulant effects on plasma clotting time and platelet aggregation examined. E456 expressed almost the same anticoagulant activities as D123 which contains six consecutive EGF-like structures of thrombomodulin. It was concluded that E456 is the minimum functional domain for both protein C-activating cofactor activity and anticoagulant activity.     

Application of ozone disinfection to remove Enterococcus seriolicida, Pasteurella piscicida, and Vibrio anguillarum from seawater.

Survival of bacterial fish pathogens, including Enterococcus seriolicida, Vibrio anguillarum, and Pasteurella piscicida, in ozonated seawater was determined in a batch system. Bacterial counts of all fish pathogens decreased at more than 0.040 to 0.060 mg of total residual oxidants (TROs) per liter, whereas no decrease in viable counts was observed at less than 0.018 to 0.028 mg of TROs per liter. The 99% inactivation point was achieved at concentrations of 0.111 mg/liter for E. seriolicida, 0.063 mg/liter for P. piscicida, and 0.064 mg/liter for V. anguillarum within 1 min. Moreover, the mean 99 and 99.9% killing concentration-contact time (C.t) products were 0.123 and 0.186 mg.min/liter for E. seriolicida, 0.056 and 0.084 mg.min/liter for P. piscicida, and 0.081 and 0.123 mg.min/liter for V. anguillarum, respectively. However, the mean 99 and 99.9% C.t products for the mixed population in coastal seawater were 0.200 and 0.621 mg.min/liter. These results strongly suggest that ozone treatment at more than 1.0 mg of TROs per liter for several minutes is able to disinfect seawater for mariculture efficiently.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Prolonged circulation time in vivo of large unilamellar liposomes composed of distearoyl phosphatidylcholine and cholesterol containing amphipathic poly(ethylene glycol).

The effect of poly(ethylene glycol) (PEG) on the circulation time of liposomes in mice was examined by employing amphipathic PEGs (phosphatidylethanolamine (PE) derivatives of PEG) with average molecular weights of 1000, 2000, 5000 and 12,000. The activity of dioleoyl phosphatidylethanolamine-PEG (DOPE-PEG) in prolonging the circulation time of egg phosphatidylcholine/cholesterol large unilamellar liposomes (ePC/CH LUVs) (200 nm) was proportional to the molecular weight of PEG, i.e., 12000 = 5000 greater than 2000 greater than 1000. On the other hand, inclusion of distearoylphosphatidylethanolamine-PEG (DSPE-PEG) or dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG) of low molecular weight such as 1000 and 2000 in distearoylphosphatidylcholine (DSPC)/CH LUVs or dipalmitoyl phosphatidylcholine (DPPC)/CH LUVs effectively increased their blood circulation time. At least 3 mol% of amphipathic PEG in liposomes was required for activity. Addition of CH, which has a bilayer-tightening effect, to DSPC/CH/DSPE-PEG2000 LUVs further increased the blood residence time. A size of less than 300 nm was essential for prolonging the residence time of amphipathic PEG-containing liposomes in blood. DSPC/CH/DSPE-PEG2000 LUVs (1:1:0.13, m/m) containing 6 mol% of PEG and 200 nm in diameter remained in the circulation for over 24 h after injection and may be clinically useful for sustained release of an entrapped drug in the bloodstream and for drug accumulation in solid tumors.